The region of periaqueductal gray (PAG) is a major component of a powerful inhibitory system that can be activated though decending forebrain imputs - the lateral hypothalamus - pre optic regions and frontal cortex. Currently information about anatomical and functional properties of the interaction between these sites and PAG and the neuron network within PAG that is involved in integration of these signals is not known. The overall goal of this project is to obtain this information. New findings in the previous funding period have led to the formulation of a novel hypotheses of the functional organization of PAG: We suggest that forebrain afferents terminate in discrete rostrocaudal "input columns". These "input columns" overlap and activate "local peptidergic networks" which integrates these signals and produce long-lasting actions on "output columns", that consists of rostrocaudally continuous columns of neuros that project out of PAG. To test this hypothesis, two interrelated series of anatomical and physiological experiments will be conducted: (A) Forebrain modulation of PAG The anatomical experiments in the first project are designed to: 1) determine the percise location of the terminals of forebrain afferents to the PAG by using anterograde and retrograde tracing procedures, 2) to obtain information about the neurotransmitters that may be involved in the interaction between forebrain and PAG by using immunocytochemistry and double labeling precedures. Parallel physiological experiments using single unit recording, iontophoresis and micropressure injection procedures will be used to determine the involvement of neurotransmitters, characterized in anatomical experiments, in the interaction between each of the above forebrain sites and PAG. B) PAG Local Networks In the second project, PAG slices maintained in vitro will be used Pharmacological experiments using a novel microstimulation technique, single unit recording and micropressure injection of neuropeptides will be conducted to obtain information about the functional characteristics of PAG local networks and the role of neuropeptides and their functions. Information obtained in these studies will increase our understanding of the properties of the mediated pain inhibitory system.